Project Summary Mitochondrial DNA (mtDNA) encodes RNAs and proteins critical for cell function. However, pathways that control mtDNA segregation and copynumber in mammalian cells are not well understood. The goals of this work are to identify and characterize the unknown protein machinery that segregates replicating mtDNAs into discrete nucleoid structures at ER-mitochondria contact sites and to describe the mechanisms and pathways that regulate mtDNA copy number. The proposed experiments will provide fundamental insight into the mechanism of mtDNA segregation and copynumber control, and how those processes are regulated, potentially leading to the discovery and characterization of novel pathways that regulate the inheritance of mtDNA disease haplotypes. In Aim 1, cutting-edge live-cell microscopy, proteomics, and high-throughput sequencing technologies will be employed to dissect the molecular functions of mtDNA segrosome candidate proteins identified in preliminary experiments. Aim 2 will address which cellular pathways are critical to regulation of mtDNA copy number in human cells. This will be accomplished by systematic genome-wide screens for modulators of mtDNA depletion recovery, single-cell RNA sequencing to cluster genes by their transcriptional responses to mtDNA loss, and the characterization of candidate mtDNA copynumber effector proteins in human cells depleted of mtDNA. These experiments will provide fundamental insight into the mechanism of mtDNA segregation and copynumber control, and how those processes are regulated, potentially leading to the discovery and characterization of novel pathways that regulate the inheritance of mtDNA disease haplotypes.